US20220276230A1 - Reagent card, detection method and application - Google Patents

Reagent card, detection method and application Download PDF

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Publication number
US20220276230A1
US20220276230A1 US17/627,614 US202017627614A US2022276230A1 US 20220276230 A1 US20220276230 A1 US 20220276230A1 US 202017627614 A US202017627614 A US 202017627614A US 2022276230 A1 US2022276230 A1 US 2022276230A1
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Prior art keywords
test
hole
sample
bulge
testing liquid
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US17/627,614
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English (en)
Inventor
Jing-Feng Huang
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Guangdong Seinda Biomedical Corp
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Guangdong Seinda Biomedical Corp
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Assigned to GUANGDONG SEINDA BIOMEDICAL CORPORATION reassignment GUANGDONG SEINDA BIOMEDICAL CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HUANG, JING-FENG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces

Definitions

  • the present invention relates to a test stick, and in particular to a test stick for precise and simple in-vitro testing of a small amount of sample, and a testing method and application thereof.
  • Point-of-care testing refers to a new method of performing clinical testing and bedside testing beside a patient to quickly obtain a testing result, which is usually not necessarily performed by a clinical technologist, but is to analyze immediately at a sampling location, thereby omitting complex processing procedures for specimens in laboratory testing.
  • Test sticks are commonly used tools in point-of-care testing for testing biochemical substances in blood, urine, feces and other body fluids, for example, bilirubin and biomarkers, such as protein, glucose, heme, ketone bodies, nitrite and urobilinogen, in serum, plasma and urine.
  • bilirubin and biomarkers such as protein, glucose, heme, ketone bodies, nitrite and urobilinogen, in serum, plasma and urine.
  • a test stick is composed of a stick shell (a top cover and a bottom cover) and a test strip in the stick shell.
  • the test strip is fixed in a clamp slot in the bottom cover, and the top cover and the bottom cover are combined to form a test stick.
  • the current rapid testing technology generally requires a sample volume of at least 10 uL. However, in clinical practice, some samples, such as a tear fluid sample, can be obtained only 1 uL or less.
  • the sensitivity of the existing rapid testing technology is generally 0.05 ng/mL (0.1 ng/mL for fluorescence immunoassay, and 0.05 ng/mL for quantum dot testing), and the precision is generally 5% to 15%. If calculated with the lower limit of testing of the total amount of the analyte of about 0.5 pg, the testing is difficult for a small amount of sample.
  • the present invention provides a test stick which, by means of a special design of a stick shell structure, controls the flow direction of a testing liquid on a test strip and the speed of chromatography to achieve the purpose of precise testing of small amount of samples in clinic so as to solve the problems existing in the prior art.
  • the present invention provides a test stick, comprising a top cover, a bottom cover and a test strip, the test strip being fixed by means of the top cover and the bottom cover,
  • a clamp slot structure and a bulge stage are provided inside the bottom cover;
  • the clamp slot structure is internally provided with a support bulge, with the height of the support bulge being less than or equal to the height of the bulge stage;
  • the top cover is provided with fixing plates, a reaction window, a sample loading hole and a testing liquid hole; the reaction window, the sample loading hole and the testing liquid hole are arranged in sequence; the fixing plates are distributed on two sides of the reaction window; and
  • test strip is loaded in the clamp slot structure of the bottom cover and is fixed, the test strip is supported by the bulge stage and the support bulge, and after the top cover and the bottom cover are engaged by means of a positioning hole and a positioning post, under the pressing of the fixing plates and the testing liquid hole of the top cover, the middle of the test strip bulges upward.
  • the test strip comprises a three-layer or four-layer structure, in which a first layer is a bottom plate, a second layer is a reaction membrane, and a third layer is an absorbent pad, a conjugate pad and a buffer pad; or a first layer is a bottom plate, a second layer is a reaction membrane and a buffer pad, and a third layer is a absorbent pad and a conjugate pad; or a first layer is a bottom plate, a second layer is a reaction membrane, a third layer is a absorbent pad and a conjugate pad, and a fourth layer is a buffer pad;
  • the reaction membrane is arranged in the middle of the bottom plate, a test line T and a control line C are distributed on the reaction membrane, and the test line T and the control line C correspond to the reaction window;
  • the conjugate pad is arranged at one end of the reaction membrane, the absorbent pad is arranged at the other end of the reaction membrane, and the conjugate pad corresponds to the sample loading hole;
  • the buffer pad is arranged on or under the conjugate pad, or the conjugate pad is butted with the buffer pad, and the buffer pad corresponds to the testing liquid hole.
  • a number of the clamp slot structure is two and the two clamp slot structures are distributed on two sides of the bulge stage.
  • the clamp slot structure is internally provided with 1-5 support bulges.
  • the clamp slot structure is internally provided with 3 support bulges.
  • flow guide ports are symmetrically arranged on two sides of the clamp slot structure corresponding to the testing liquid hole.
  • the depth of the clamp slot structure is 1.2-1.7 mm
  • the height of the support bulge is 0.1-0.15 mm
  • the height of the bulge stage is 0.15-0.25 mm.
  • a number of the fixing plates is four, the fixing plates comprise a first fixing plate for fixing the test strip, a second fixing plate for fixing the test strip, a third fixing plate for fixing a combination of the absorbent pad and the reaction membrane on the test strip, and a fourth fixing plate for fixing a combination of the reaction membrane and the conjugate pad on the test strip; and the fourth fixing plate is arranged between the sample loading hole and the reaction window, and the first fixing plate, the second fixing plate and the third fixing plate are distributed on the other side of the reaction window.
  • the first fixing plate, the second fixing plate, the third fixing plate and the fourth fixing plate have a width of 3-5 mm; and the first fixing plate and the second fixing plate have a height of 1.0-1.5 mm, and the third fixing plate and the fourth fixing plate have a height of 0.1-0.3 mm.
  • the sample loading hole is of a funnel-shaped structure
  • the front-side circumferential diameter of the top cover is 4-6 mm
  • the inside circumferential diameter of the top cover is 3-5 mm
  • the height of the bulge is 0.1-0.2 mm
  • an included angle between a hole wall and a horizontal plane is 30° to 45°.
  • the testing liquid hole is of a funnel-shaped structure
  • the front-side circumferential diameter of the top cover is 6-8 mm
  • the inside circumferential diameter of the top cover is 1-3 mm
  • the height of the bulge is 0.3-0.5 mm
  • the included angle between the hole wall and the horizontal plane is 30° to 45°.
  • the reaction window is of an elliptical ring-shaped structure
  • the length of the front-side ellipse of the top cover is 150-200 mm and the width thereof is 70-90 mm
  • the inside circumferential diameter of the top cover is 130-150 mm
  • the height of the bulge is 0.1-0.2 mm
  • the included angle between the hole wall and the horizontal plane is 30° to 45°
  • a contact surface between the bottom of the reaction window and the test strip is a vertical bulge of 0.01-0.02 mm.
  • the top cover comprises a hand-held end, with the surface of the hand-held end being of a threaded or embossed bulge structure.
  • the top cover and the bottom cover are engaged by means of the positioning hole and the positioning post.
  • test lines T are distributed on the reaction membrane.
  • the present application further provides a testing method using any test stick described above:
  • Step (1) Loading a Sample into a Sample Loading Hole
  • the sample is loaded onto a conjugate pad in the sample loading hole, the conjugate pad being provided with antibodies coupled to nanospheres, i.e., antibody nanospheres, and analyte in the sample is immunologically bound with the antibody nanospheres on the conjugate pad to form antibody-nanosphere complexes; and
  • Step (2) Loading a Testing Liquid into a Testing Liquid Hole
  • chromatography of the testing liquid is started in a direction of an absorbent pad end of the test strip, and the testing liquid under chromatography passes through the position of the sample loading hole to dissolve the antibody nanospheres on the conjugate pad that are not bound to the analyte and the antibody-nanosphere complexes that have been bound to the analyte; the chromatography of the testing liquid on the test strip undergoes “upslope, parallel and downslope”; and the testing liquid moves on the test strip and undergoes a second immune recognition reaction in the “parallel” stage, in which the antibody-nanosphere complexes in step (1) undergo the second immune recognition reaction at the test line T and begin to develop color, while the antibody nanospheres that do not react with the test line T continue to move forward and bind to the control line C to develop color.
  • 0.1-50 ul of the sample is loaded into the sample loading hole; and 0-150 ul of the testing liquid is loaded into the testing liquid hole.
  • the concentration of the analyte in the sample is calculated by measuring signal values of the T line and the C line with an instrument or by comparing color depths of the T line and the C line with a color card.
  • the testing method described above is used for testing of small amount of samples, such as tear fluid, wound exudate, tissue fluid, sweat, aqueous humor and ocular surface lotion, can also be used for testing of routine samples, such as blood (peripheral blood, serum, plasma), saliva and urine, and can also be used for testing of other non-clinical samples, for example, for testing in pets, animal husbandry, agriculture, fish, food, environmental protection, etc.
  • samples such as tear fluid, wound exudate, tissue fluid, sweat, aqueous humor and ocular surface lotion
  • routine samples such as blood (peripheral blood, serum, plasma), saliva and urine
  • other non-clinical samples for example, for testing in pets, animal husbandry, agriculture, fish, food, environmental protection, etc.
  • the height of the support bulge is less than or equal to the height of the bulge stage, after the test strip is loaded in the clamp slots and is fixed, the test strip is supported by the support bulges in the clamp slots at two ends to form a “bridge” structure.
  • the fixing plates, the sample loading hole and the testing liquid hole on the top cover cooperate with the bulge stage to press downward, such that the middle portion of the reaction membrane bulges upward to form an “arch-bridge” structure with the horizontal plane.
  • FIG. 1 is a schematic view of a top cover
  • FIG. 2 is a schematic view of a bottom cover
  • FIG. 3 a is a schematic view of example 1 of a test strip
  • FIG. 3 b is a schematic view of example 2 of a test strip
  • FIG. 3 c is a schematic view of example 3 of a test strip
  • FIG. 4 is a schematic view of a test stick
  • FIG. 5 a is a schematic front view of the top cover
  • FIG. 5 b is a schematic reverse view of the top cover
  • FIG. 5 c is a schematic cross-sectional view of the top cover
  • FIG. 6 a is a schematic front view of the bottom cover
  • FIG. 6 b is a schematic reverse view of the bottom cover
  • FIG. 6 c is a schematic cross-sectional view of the bottom cover
  • FIG. 7 a is a schematic exploded view of the test stick
  • FIG. 7 b is a schematic cross-sectional view of the test stick
  • FIG. 8 a is a schematic cross-sectional view of the test stick after assembly
  • FIG. 8 b is a partial enlarged cross-sectional view of the test stick after assembly.
  • Embodiments of the present invention provide a test stick to solve the problems exiting in the prior art.
  • test stick provided by the present invention will be specifically described below in conjunction with the accompanying drawings.
  • the present invention provides a test stick, comprising a top cover, a bottom cover and a test strip.
  • the top cover and the bottom cover are engaged by means of positioning holes and positioning posts so as to fix the test strip.
  • the top cover is provided with positioning posts G 01
  • the bottom cover is provided with positioning holes D 01
  • the test strip is fixed by means of cooperation of the positioning posts G 01 and the positioning holes D 01 .
  • the positioning posts G 01 are symmetrically distributed in three groups in position, including a front, a middle and a rear group, and the positioning posts G 01 have a height of 2.5-3.5 mm and a diameter of 1.0-1.5 mm, and mainly support and fix the test stick.
  • the positioning holes D 01 are symmetrically distributed in three groups in position, including a front, a middle and a rear group, and post holes have a height of 2.5-3.5 mm and a diameter of 1.1-1.6 mm, and cooperate with the posts of the top cover to support and fix the test stick.
  • clamp slot structures (D 02 , D 06 ) and a bulge stage D 04 are provided inside the bottom cover.
  • the clamp slot structure is internally provided with a support bulge D 03 , with the height of the support bulge D 03 being less than or equal to the height of the bulge stage D 04 .
  • the top cover is provided with fixing plates (G 02 , G 03 , G 04 , G 06 ), a reaction window G 05 , a sample loading hole A and a testing liquid hole B.
  • the reaction window G 05 , the sample loading hole A and the testing liquid hole B are arranged in sequence.
  • the fixing plates (G 02 , G 03 , G 04 , G 06 ) are distributed on two sides of the reaction window G 05 .
  • test strip After the test strip is loaded in the clamp slot structures (D 02 , D 06 ) of the bottom cover and is fixed, the test strip is supported by the bulge stage D 04 and the support bulge D 03 , and after the top cover and the bottom cover are engaged by means of the positioning holes and the positioning posts, under the pressing of the fixing plates (G 02 , G 03 , G 04 , G 06 ) and the testing liquid hole B of the top cover, the middle of the test strip bulges upward.
  • the test strip comprises a three-layer or four-layer structure, in which a first layer is a bottom plate 1 , with the material thereof being preferably PVC, a second layer is a reaction membrane 4 , and a third layer is an absorbent pad 7 , a conjugate pad 3 and a buffer pad 2 ; or the first layer is the bottom plate 1 , the second layer is the reaction membrane 4 , the third layer is the absorbent pad 7 and the conjugate pad 3 , and the fourth layer is the buffer pad 2 .
  • the reaction membrane 4 is arranged in the middle of the bottom plate 1 , a test line T 5 and a control line C 6 are distributed on the reaction membrane 4 , and the test line T 5 and the control line C 6 correspond to the reaction window G 05 . There may be two test lines T 5 for distinguishing between two diseases.
  • the conjugate pad 3 is arranged at one end of the reaction membrane 4 , the absorbent pad 7 is arranged at the other end of the reaction membrane 4 , and the conjugate pad 3 corresponds to the sample loading hole A.
  • the buffer pad 2 is arranged on the conjugate pad 3 , or the conjugate pad 3 is butted with the buffer pad 2 , and the buffer pad 2 corresponds to the testing liquid hole B.
  • Two clamp slot structures are provided and are respectively a first clamp slot structure D 02 and a second clamp slot structure D 06 , and the first clamp slot structure D 02 and the second clamp slot structure D 06 are distributed on two sides of the bulge stage D 04 .
  • the clamp slot structure is internally provided with 1-5 support bulges.
  • the clamp slot structure is internally provided with 3 support bulges, as shown in FIGS. 6A-6C , the first clamp slot structure D 02 and the second clamp slot structure D 06 are each internally provided with three support bulges D 03 .
  • Flow guide ports D 05 are symmetrically arranged on two sides of the second clamp slot structure D 06 corresponding to the testing liquid hole B.
  • the two groups of flow guide ports D 05 are symmetrically arranged at two ends of the clamp slot, which ensures that the testing liquid undergoes chromatography along the test strip so as to prevent the liquid from sticking to the stick shell or seeping to the back of the test strip due to the surface tension of the test stick.
  • the first clamp slot structure D 02 and the second clamp slot structure D 06 have a width of 3-5 mm, a length of 10-18 mm, and a depth of 1.2-1.7 mm, the support bulge D 03 has a height of 0.1-0.15 mm and a width of 3-5 mm, and the bulge stage D 04 has a height of 0.15-0.25 mm.
  • the support bulges D 03 are similar to the working principle of bridge piers, are distributed at both ends of the clamp slot and provided as three at the absorbent pad end, and act to fix and support the test strip.
  • the fourth fixing plate G 06 is arranged between the sample loading hole and the reaction window, and the first fixing plate G 02 , the second fixing plate G 03 and the third fixing plate G 04 are distributed on the other side of the reaction window G 05 .
  • the first fixing plate G 02 , the second fixing plate G 03 , the third fixing plate G 04 and the fourth fixing plate G 06 have a width of 3-5 mm.
  • the first fixing plate G 02 and the second fixing plate G 03 have a height of 1.0-1.5 mm, and the third fixing plate G 04 and the fourth fixing plate G 06 have a height of 0.1-0.3 mm.
  • the first fixing plate G 02 and the second fixing plate G 03 are used to fix the test strip after the assembly of the stick shell, which is different from the test strips of other manufacturers which are fixed with tapes.
  • the third fixing plate G 04 has a main function to fix a combination of the absorbent pad 7 and the reaction membrane 4 on the test strip, which ensures that there is traction force for the liquid to continue chromatography and movement in the later stage of immunochromatography.
  • the fourth fixing plate G 06 has a main function to fix a combination of the conjugate pad 3 and the reaction membrane 4 on the test strip, such that the reactant of the first step and the solution reach the reaction membrane 4 smoothly to carry out the reaction of the second step.
  • the sample loading hole A is of a funnel-shaped structure, the front-side circumferential diameter of the top cover is 4-6 mm, the inside circumferential diameter of the top cover is 3-5 mm, the height of the bulge is 0.1-0.2 mm, and an included angle between a hole wall and a horizontal plane is 30° to 45°.
  • the wide-mouth design is conducive to contact with the conjugate pad 3 of the test strip when a small amount of sample is loaded, and is also convenient for a variety of instruments to load a sample after the sample is collected, so as to enable the sample to be fully absorbed by the conjugate pad.
  • the testing liquid hole B is of a funnel-shaped structure, the front-side circumferential diameter of the top cover is 6-8 mm, the inside circumferential diameter of the top cover is 1-3 mm, the height of the bulge is 0.3-0.5 mm, and the included angle between the hole wall and the horizontal plane is 30° to 45°.
  • the liquid is stored after the testing liquid is loaded, and a hydraulic pressure is formed to drive the liquid to move toward the conjugate pad 3 and the reaction membrane 4 .
  • the bulge having a height of 0.3-0.5 mm is combined with the bottom cover and then tightly presses the buffer pad 2 at the clamp slot position, with the volume of about 90 ⁇ L, and releases the liquid directionally and slowly under a capillary action, so as to prevent the insufficient first-step immune reaction due to too fast liquid flow rate.
  • the reaction window G 05 is of an elliptical ring-shaped structure, the length of the front-side ellipse of the top cover is 150-200 mm and the width thereof is 70-90 mm, the inside circumferential diameter of the top cover is 130-150 mm, the height of the bulge is 0.1-0.2 mm, the included angle between the hole wall and the horizontal plane is 30° to 45°, and a contact surface between the bottom of the reaction window and the test strip is a vertical bulge of 0.01-0.02 mm.
  • the design of the vertical bulge of 0.01-0.02 mm prevents the liquid from being affected by an edge effect of an inward bulge during chromatographic movement of the liquid on the reaction membrane 4 .
  • the bulge has a height of 0.1-0.2 mm, which is the same as the height of the sample loading hole A, both being 0.3-0.5 mm lower than the testing liquid B, so as to ensure the air circulation inside the test stick, so that the liquid chromatography process is not affected by the air pressure.
  • the reaction window G 05 is a position where the second step of the immune reaction is carried out. After the reaction, the concentration of the analyte in the sample can be calculated by measuring signal values of the T line and the C line with an instrument or by comparing color depths of the T line and the C line with a color card.
  • the top cover comprises a hand-held end C, with the surface of the hand-held end C being of a threaded or embossed bulge structure.
  • the bulge stage D 04 has a length of 15.0-0.0 mm, a width of 3.0-5.0 mm, and a height of 0.15-0.25 mm. This bulge stage D 04 is located at the reaction window of the top cover, and is a position where the second step of the immune binding reaction is carried out. Since the height of the support bulge D 03 is less than or equal to the height of the bulge stage D 04 , after the test strip is loaded in the clamp slots and is fixed, the test strip is supported by the support bulges D 03 in the clamp slots at two ends to form a “bridge” structure.
  • the fixing plates (G 02 , G 03 , G 04 , G 06 ), the sample loading hole A and the testing liquid hole B on the top cover cooperate with the bulge stage D 04 to press downward, such that the horizontal plane of the reaction membrane 4 in the middle of the test strip is raised, and the reaction membrane 4 in the middle bulges upward by 0.15-0.25 mm and forms an “arch-bridge” structure with the horizontal plane (see FIG. 8 b ).
  • the formation of this “arch-bridge” structure plays an important role in the accuracy and precision of the testing result of the test stick. This design has the following two effects: I.
  • control of the speed of chromatography to further expand the role of molecular sieve of the reaction membrane in the chromatography process of the liquid, the horizontal plane of the test strip gradually rises.
  • the liquid is in an upslope state during chromatography, and at this time, the speed of chromatography is decreased to prolong the time of the second step of reaction, such that the analyte that is not fully bound in the first step is further recognized and bound in the process of movement so as to, in the case of decreasing the speed of chromatography, further exert the role of molecular sieve of the reaction membrane to separate non-targets from some samples; and after the second step of sufficient reaction, the liquid begins to be in a downslope state, and under the action of the absorbent pad, the speed of chromatography is increased, the entire reaction process is completed within 10-15 min, and the speed of chromatography of liquid of the test stick is about 45 mm/s.
  • the reagent strip forms an “arch-bridge” structure (see FIG. 8 b ), and the liquid performs chromatographic movement in the same direction under the capillary action so as to prevent the loss of analyte due to the liquid spreading around caused by hydraulic pressure when the testing liquid is dripped, which ensures the effective testing concentration of the small amount of sample liquid.
  • the present application further provides a testing method using any test stick described above:
  • Step (1) Loading a Sample into a Sample Loading Hole
  • the sample is loaded onto a conjugate pad in the sample loading hole, the conjugate pad being provided with antibodies coupled to nanospheres, i.e., antibody nanospheres, and analyte in the sample is immunologically bound with the antibody nanospheres on the conjugate pad to form antibody-nanosphere complexes; and
  • Step (2) Loading a Testing Liquid into a Testing Liquid Hole
  • chromatography of the testing liquid is started in a direction of an absorbent pad end of the test strip, and the testing liquid under chromatography passes through the position of the sample loading hole to dissolve the antibody nanospheres on the conjugate pad that are not bound to the analyte and the antibody-nanosphere complexes that have been bound to the analyte; the chromatography of the testing liquid on the test strip undergoes “upslope, parallel and downslope” (see FIG.
  • the testing liquid moves on the test strip and undergoes a second immune recognition reaction in the “parallel” stage, in which the antibody-nanosphere complexes in step (1) undergo the second immune recognition reaction at the test line T and begin to develop color, while the antibody nanospheres that do not react with the test line T continue to move forward and bind to the control line C to develop color.
  • 0.1-50 ul of the sample is loaded into the sample loading hole; and 0-150 ul of the testing liquid is loaded into the testing liquid hole.
  • the concentration of the analyte in the sample is calculated by measuring signal values of the T line and the C line with an instrument or by comparing color depths of the T line and the C line with a color card. Whether the color development of the C line is sufficient is an important indicator to evaluate whether the reaction of this test stick is complete and whether the measured value is accurate.
  • the C line is a control line, which plays an important role in the interpretation of the testing result of the test stick. The C line needs to be fully developed and reach a predetermined value before the testing result is determined to be valid. If the C line is not developed, the measured value is low, there may be interference, and the testing result is invalid. If the C line is developed insufficiently, the measured value is low, indicating that the user may operate incorrectly and the testing result is invalid.
  • the testing method described above is used for testing of a tear fluid, wound exudate, tissue fluid, sweat, aqueous humor, and ocular surface lotion.
  • the front side (the top cover) of the test stick is designed to have a sample loading hole A and a testing liquid hole B.
  • sample volume is 1-5 ⁇ L
  • the testing liquid is dripped into the testing liquid hole B, and then the reaction of immunochromatography is started.
  • the whole process is simple to operate.
  • the sample loading hole A is located at the conjugate pad of the test strip.
  • the conjugate pad is made of hydrophilic glass cellulose, and the conjugate pad is provided with nanoparticle-labeled antibody AB-1.
  • the sample is directly loaded to the conjugate pad, and the hydrophilic material is conducive to the diffusion of the sample, so that the analyte in the sample is recognized and immunologically bound with the nanoparticle-labeled antibody AB-1.
  • the utilization rate of the sample is almost 100%, and a small amount of liquid of 0.1-5 ⁇ L will not cause the subsequent chromatography reaction.
  • a larger volume of sample ⁇ 50 ⁇ L, can be loaded directly for testing.
  • the analyte binds to the nanosphere-labeled antibody AB-1 to form the bottom and middle layers of a “sandwich” structure of the double antibody sandwich method, so as to complete the first step of immunochromatography testing.
  • the testing liquid After the testing liquid is loaded into the testing liquid hole B, the testing liquid begins chromatography in a direction of the absorbent pad end of the test strip. From the beginning to the completion of the reaction, the chromatography of the liquid on the test strip undergoes “upslope, parallel and downslope” (see FIG. 8 b ), and the control of the flow direction and flow rate of the liquid depends on the special design of the test stick.
  • the liquid moves on the test strip and performs the second immune recognition reaction in the “parallel” stage, so as to form a complete “sandwich” structure with the test line T and begin to develop color, with the depth of the color being positively correlated with the content of the analyte, i.e., the “sandwich” middle layer.
  • the nanoparticles that do not react with the T line continue to move forward, and are bound to the control line C to develop color.
  • the concentration of the analyte in the sample can be calculated by measuring signal values of the T line and the C line with an instrument or by comparing color depths of the T line and the C line with a color card. Whether the color development of the C line is sufficient is an important indicator to evaluate whether the reaction of this test stick is complete and whether the measured value is accurate.
  • the flow direction of a testing liquid on the test strip and the speed of chromatography are controlled to achieve the purpose of accurately testing a small amount of sample in clinic, which fills up a gap in the market of existing POCT testing products for small amount of samples.
  • test stick provided in the present application can be used for the auxiliary diagnosis of dry eye:
  • Step (1) Loading a Sample into a Sample Loading Hole
  • a tear fluid sample is collected, 1 ⁇ L of the tear fluid sample is loaded onto a conjugate pad in the sample loading hole, the conjugate pad being provided with a-lymphotoxin (LTA) antibodies coupled to nanospheres, i.e., antibody nanospheres, and analyte in the sample is immunologically bound with the antibody nanospheres on the conjugate pad to form antibody-nanosphere complexes; and
  • LTA -lymphotoxin
  • Step (2) Loading a Testing Liquid into a Testing Liquid Hole
  • chromatography of the testing liquid is started in a direction of an absorbent pad end of the test strip, and the testing liquid under chromatography passes through the position of the sample loading hole to dissolve the antibody nanospheres on the conjugate pad that are not bound to the analyte and the antibody-nanosphere complexes that have been bound to the analyte; the chromatography of the testing liquid on the test strip undergoes “upslope, parallel and downslope” (see FIG.
  • LTA a-lymphotoxin
  • the testing liquid moves on the test strip and undergoes a second immune recognition reaction in the “parallel” stage, in which the antibody-nanosphere complexes in step (1) undergo the second immune recognition reaction at the test line T and begin to develop color, while the antibody nanospheres that do not react with the test line T continue to move forward and bind to the control line C to develop color.
  • the sensitivity reached 0.15 ng/mL
  • the precision CV reached 5% to 10%.
  • test stick provided in the present application can be used for the auxiliary diagnosis of inflammation:
  • Step (1) Loading a Sample into a Sample Loading Hole
  • MMP-9 matrix metalloproteinase 9
  • Step (2) Loading a Testing Liquid into a Testing Liquid Hole
  • MMP-9 matrix metalloproteinase 9
  • chromatography of the testing liquid is started in a direction of an absorbent pad end of the test strip, and the testing liquid under chromatography passes through the position of the sample loading hole to dissolve the antibody nanospheres on the conjugate pad that are not bound to the analyte and the antibody-nanosphere complexes that have been bound to the analyte; the chromatography of the testing liquid on the test strip undergoes “upslope, parallel and downslope” (see FIG.
  • MMP-9 matrix metalloproteinase 9
  • the testing liquid moves on the test strip and undergoes a second immune recognition reaction in the “parallel” stage, in which the antibody-nanosphere complexes in step (1) undergo the second immune recognition reaction at the test line T and begin to develop color, while the antibody nanospheres that do not react with the test line T continue to move forward and bind to the control line C to develop color.
  • the sensitivity reached 1.0 ng/mL
  • the precision CV reached 5% to 10%.
  • test stick provided in the present application can be used for the auxiliary diagnosis of allergic conjunctivitis:
  • Step (1) Loading a Sample into a Sample Loading Hole
  • ocular surface lotion sample 2.2 ⁇ L of the ocular surface lotion sample is loaded onto a conjugate pad in the sample loading hole, the conjugate pad being provided with total immunoglobulin E (IgE) antibodies coupled to nanospheres, i.e., antibody nanospheres, and analyte in the sample is immunologically bound with the antibody nanospheres on the conjugate pad to form antibody-nanosphere complexes; and
  • IgE total immunoglobulin E
  • Step (2) Loading a Testing Liquid into a Testing Liquid Hole
  • chromatography of the testing liquid is started in a direction of an absorbent pad end of the test strip, and the testing liquid under chromatography passes through the position of the sample loading hole to dissolve the antibody nanospheres on the conjugate pad that are not bound to the analyte and the antibody-nanosphere complexes that have been bound to the analyte; the chromatography of the testing liquid on the test strip undergoes “upslope, parallel and downslope” (see FIG.
  • IgE immunoglobulin E
  • the testing liquid moves on the test strip and undergoes a second immune recognition reaction in the “parallel” stage, in which the antibody-nanosphere complexes in step (1) undergo the second immune recognition reaction at the test line T and begin to develop color, while the antibody nanospheres that do not react with the test line T continue to move forward and bind to the control line C to develop color.
  • the sensitivity reached 0.5 IU/mL
  • the precision CV reached 5% to 10%.
  • test stick provided in the present application can be used for the auxiliary diagnosis of conjunctivitis and the differential diagnosis of conjunctivitis caused by allergy or viral infection:
  • Step (1) Loading a Sample into a Sample Loading Hole
  • ocular surface lotion sample After an ocular surface lotion sample is collected, 2.2 ⁇ L of the ocular surface lotion sample is loaded onto a conjugate pad in the sample loading hole, the conjugate pad being provided with total immunoglobulin E (IgE) antibodies and virus antibodies both coupled to nanospheres, i.e., antibody nanospheres, and analyte in the sample is immunologically bound with the antibody nanospheres on the conjugate pad to form antibody-nanosphere complexes; and
  • IgE total immunoglobulin E
  • Step (2) Loading a Testing Liquid into a Testing Liquid Hole
  • chromatography of the testing liquid is started in a direction of an absorbent pad end of the test strip, and the testing liquid under chromatography passes through the position of the sample loading hole to dissolve the antibody nanospheres on the conjugate pad that are not bound to the analyte and the antibody-nanosphere complexes that have been bound to the analyte; the chromatography of the testing liquid on the test strip undergoes “upslope, parallel and downslope” (see FIG.
  • the testing liquid moves on the test strip and undergoes a second immune recognition reaction in the “parallel” stage, in which the antibody-nanosphere complexes in step (1) undergo the second immune recognition reaction at the two test lines T (respectively being a virus test line T and a total IgE test line T) and begin to develop color, and according to the color development of the virus test line T and the total IgE test line T, it is possible to determine whether the eye disease is caused by viral infection or allergy, while the antibody nanospheres that do not react with the test line T continue to move forward and bind to the control line C to develop color. After multiple tests, the sensitivity reached 0.5 IU/mL or 0.5 ng/mL, and the precision CV reached 5% to 10%.

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